3alpha,20 - diacetoxy - 16beta - lower alkyl - 17(20)-oxido- pregnane-11-one and intermediate in the preparation thereof



United States Patent M 3,461,117 30;,20 DIACETOXY 16B LOWER ALKYL17(20)- OXIDO-PREGNANE-ll-ONE AND INTERNIEDI- ATE IN THE PREPARATIONTIEREOF Richard Rausser, Union, and Eugene P. Oliveto, Glen Ridge, N.J.,assignors to Schering Corporation, Bloomfield, N.J., a corporation ofNew Jersey No Drawing. Continuation-impart of application Ser. No.673,141, July 22, 1957. This application Aug. 18, 1960, Ser. No. 50,314

Int. (ll. Cil7c 173/00, 169/36 US. Cl. 26l)239.55

4 Claims This application is a continuation-in-part of application Ser.No. 673,141, filed July 22, 1957.

This invention relates to a new group of steroid intermediates and withmethods for the conversion of these intermediates into a new group ofalkylated steroid compounds. More particularly, this invention isconcerned with steroid intermediates which are useful in the preparationof 16/3-alkyl-A -pregnenes and their n -analogs.

We have found that the l6fi-alkylated steroid compounds, andparticularly the 16 3-methylated steriod compounds, which are preparedfrom the novel intermediates of this invention are valuable in thetreatment of inflammatory diseases such as arthritis. These new 16,6-alkylated steroid compounds are represented by formulae A and B on page9 and are disclosed in our copending applications Ser. Nos. 673,141 nowabandoned, filed July 22, 1957, and 733,843, now Pat. No. 3,164,618filed May 8, 1958, of which the instant application is a continuation.In said formulae, X may be 0, (H, aOH), or (H, ,SOH); R may be H or acarboxylic acyl radical (preferably one containing from 1-8 carbon atomssuch as, for example, a lower alkanoyl radical, e.g. acetate,propionate, benzoate, phthalate, dimethyl acetate, trimethyl acetate,tert.-butyl acetate, phenoxy acetate, thiophene carboxylate, nicotinate,and the like), and R is a lower alkyl group such as, for example,,B-rnethyl, B-ethyl, ,B-propyl, fi-isopropyl, fl-butyl, fi-terL-butyl,and the like. Those compounds wherein X represents (H, 0H), while nottherapeutically active per se, are readily converted by known oxidativetechniques into ll-keto compounds which possess physiological activity.Particularly preferred are those compounds wherein X is O or (H, ,BOH);R is the acetate radical and R is the methyl group; the A -c0mpoundsbeing the more potent.

The addition of the 16B-alkyl group appears to prolong the duration ofaction of the steroid over that shown by their non-methylated analogs.In addition, the

3,461,117 Patented Aug. 12, 1969 16,6-alkylation of steroids possessinganti-inflammatory properties confers on the parent steroids the propertyof naturesis and diuresis. As a result, steroid compounds which possessanti-inflammatory properties, but which are limited in their usefulnessin the treatment of disease because of their inherent sodium retainingeffects, have their usefulness extended by 16fl-alkylation. It is clear,therefore, that the discovery of steroid intermediates which are readilyconvertible to the 16,6-alkylated steroid compounds discussed above is avaluable contribution to the pharmaceutical art.

Conversion of the novel intermediates of this invention into16-alkylated steroid compounds is described hereinafter in detail and isfurther illustrated by the sequence of reactions shown on pages 9, 10and 11. For purposes of illustration, merely, the preferred methyl andacetate groups are employed in the following description, as well as inthe reaction sequences, as the 16,8-a1kyl radical and the 2l-acylsubstituent, respectively. No limitation, however, is intended therebyexcept as defined by the appended claims.

As the starting material in the ultimate preparation of the16p-alkylated steroid compounds described above, we prefer to employ theeasily available substance 16- pregnene-iiu-ol-l1,20-dione-3-acylate(i.e. acetate) (I) as shown in reaction sequence A. Reaction of suchester with diazomethane gives in excellent yield, the pyrazoline {c.g.alkylidenediazo) intermediate16,17a-alkylidinediaZopregnane-Iia-ol-l1,20-dione 3-acetate (II).Compound (II) upon pyrolysis at, or above, its melting point isconverted into 16-methyl-16pregnene-3u-ol-11,20-dione 3-acetate (III).Where the 16,8-ethyl, l6fi-propyl or 16,8- butyl derivative is desired,for example, the corresponding diazoethane, diazopropane or diazobutaneis employed instead of diazomethane in the above reaction. Although weprefer to pyrolize the pyrazoline by heating above its melting point,such pyrolysis may also be effected by heating the substance in a highboiling solvent, such as p-cymene, or Tetralin and the like. Theunsaturation in the D-ring is conveniently removed by reductivehydrogenation preferably in the presence of a catalyst, such aspalladium, whereupon there is obtained16(3-methylpregnane-Bor-ol-l1,20-dione 3-acetate (IV).

In order to introduce a hydroxyl group at C-17, we have found that theC-20 keto group of IV can be easily converted into an enol-acetate byrefluxing IV with acetic anhydride and a strong acid, such asp-toluenesulfom'c acid, for example. We prefer not to isolate theenol-acetate but rather react said substance in situ with a peroxy acid,

such as peracetic acid, whereby a second intermediary nonisolatedproduct, being a 17,20-epoxide, is obtained. Treatment of the reactionmixture with alkali hydrolyzes the epoxide in situ and thereby forms16fl-methylpregnane-3a,17u-diol-l1,20-dione (V). In this step, wherebythe hydroxyl group is introduced at C-l7, other acetylating agents, suchas isopropenyl acetate, may be equally used in place of aceticanhydride. Furthermore, the strong acid catalyst is not necessarilylimited to p-toluenesulfonic acid, since other strong acids such asperchloric acid are equally effective to produce the intermediateenol-acetate. As indicated above, the enol-acetate is converted to therespective 17,20-epoxide by means of a peroxy acid, as exemplified byperacetic, perbenzoic, monoperphthalic, pertifiuoroacetic acids and thelike.

Acetoxylation of the C-21 methyl group is effected in the conventionalmanner, such as by bromination of the C-21 methyl group, followed byreaction of the bromocompound with sodium or potassium acetate, whereby165 methylpregnane 3oz,17oc,21 triol-ll,20-dione 21- acetate (VI) isformed. Although acetoxylation has been shown by way of illustration, itwill be apparent to one skilled in the art, that othercarboxyacyloxylations, such as the introduction of a propionoxy groupfor example, may analogously be effected.

In order to obtain the 3-keto-A or 3-keto-A -system, compound (V1) isfurther transformed as follows: The hydroxyl group at C-3 is convertedto a keto group, preferably by means of N-bromoacetarnide whereby VII 18produced. It is apparent that other equivalent oxidizing agents can beemployed in this step, such as N-bromosuccinimide, chromiumtrioxide-pyridine, chromium trioxideacetone-sulfuric acid mixture andthe like. The A -double bond is now conveniently introduced into theA-ring by brominating VII in the conventional manner whereby theintermediary bromo-compound (VIII) is formed, which may or may not beisolated. Although we prefer to halogenate with bromine, otherhalogenating agents such as chlorine are equally effective.Dehydrohalogenation of VIII in a conventional manner such as by reactionwith semicarbazide followed by hydrolysis of the so-formed3-mono-semicarbazone, or by refluxing with dimethylformamide optionallyin the presence of a lithium chloride, or by refluxing with organicbases such as collidine, cause elimination of hydrogen halide andinsertion of the A -double bond, yielding 16,8-methylcortisone21-acetate (IX). Compound (IX) may be saponified so as to produce thediol-trione, X, by reacting the former with hydrolytic agents such asaqueous methanolic potassium bicarbonate or with such reagents as sodiumcarbonate, sodium hydroxide, sodium alkoxides, and acids such asptoluenesulfonic acid. 16fi-methylcortisone (X) may now be re-esterifiedat C-Zl with any acid residue which is preferred. We have found that, asis generally known in the art, esterification enhances the duration ofactivity of the steroid alcohol and provides for a compound which may beadministered parenterally. We have found that using acid residues, suchas obtained from acetic, propionic, trimethylacetic, t-butylacetic,cyclopentylpropionic, furoic, phenoxyacetic and the like, provide usefulesters. Similarly, half-esters of dibasic acids, such as obtained fromphthalic, succinic, tartaric, citric and the like, provide for esterswhich can be solubilized by formation of a salt of the free carboxylgroup with an alkali metal, such as sodium. In place of the dibasicorganic acids, there may be used inorganic acids, such as phosphate andthe like, thus producing a dihydrogen phosphate ester which itself canbe further solubilized by salt formation.

Introduction of the A -bond is preferably carried out by subjecting IXor X to the microbiological action of a dehydrogenating microorganism.We prefer to utilize Bacillus sphaericus (A.T.C.C. 7055) orCorynebacterium simplex (A.T.C.C. 6946) according to analogousprocedures described in Belgian Patent No. $40,478. It is essential,however, that the compound subjected to fermentative dehydrogenationcontain at least a 3-keto-A -system and thus the first opportune time toinsert the A -bond is after the formation of compound IX or compound X.There is thus produced l6B-methyl-1,4-pregnadiene-17a,21-diOl-3,11,20-t1'IOI1t3.

The dehydrogenation of the A-ring whereby the A bond is inserted mayalso be accomplished by methods now known, such as by reacting thepregnene IX or the pregnane VII with selenium dioxide, for example, atelevated temperatures or by the well-known methods of halogenating anddehydrohalogenating.

This latter dehydrogenative procedure is exemplified by reactionsequence B whereby the pregnane VII is dihalogenated preferably withbromine, thus forming the intermediary 2,4-dibromo 16B methylpregnane-1704.21- diol-3,l1,20-trione Zl-acetate (XII). Didehydrobromination withbasic agents, preferably dimethylformamide, produces the Zl-acetate of16,8-methylprednisone (XIII). Compound XIII may be saponified so as toproduce methylprednisone (XI) by use of any of the hydrolytic agentsdescribed above. It is obvious that at the final state ofl6B-rnethylprednisone, various ester groups may be introduced at 0-21.

The foregoing reaction sequence and discussions have applied to thepreparation of compounds containing a keto function at C-11. It isapparent that if any of the starting materials contains a hydroxyl groupat C-1l, said group will be carried through the series of reactions.However, keeping in mind the availability of the original startingmaterial, 16-pregnene-3a-ol-11,20-dione 3-acetate (I), we prefer toinitially carry out the recations so as to produce ll-keto substancesand when required, reduce the ll-keto group to an ll-hydroxyl grouppreferably having the ,8 configuration. Such a transformation isoutlined in reaction sequence C whereby the 3,20-bis-semicarbazone (XIV)of 16B-methylcortisone acetate (IX) is formed from the reaction of IXwith semicarbazide. Having now protected the sensitive 3,20-diketogroups, the ll-keto function is reduced with sodium borohydride inaqueous tetrahydrofuran, thus yielding the bis-semicarbazone of16/9-methylhydrocortisone (XV). The substituted groups at C3 and C-2Oare removed in a conventional manner, for example, by means of pyruvicacid, or by hydrolytic procedures such as dilute hydrochloric acid ornitrous acid thus yielding 16,8-methylhydrocortisone (XVI).

Other methods of protecting the 3-keto groups may be employed, such asforming a 3,20-bis-ethylene ketal and then reducing the ll-keto group bymeans of sodium borohydride, lithium borohydride, or lithium aluminumhydride and the like. Acid hydrolysis of the ll-hydroxybis-ketal sofor-med yields l6fi-methyl-hydrocortisone (XVI).

lfifi-methylhydrocortisone (XVI) may be converted to16,8-methylprednisolone (XVII) by means of the microbiologicalfermentation or chemical methods described above for the conversion of16p-methylcortisone to 165- methylprednisone. Similarly,16,8-methylprednisolone (XVII) may be esterified according to any of thewellknown techniques so as to produce a 21-acetate, such as XVIII.

Alternative to the foregoing, l6fl-methylprednisone 21- acetate (XIII)itself may be converted to l6fi-methylprednisolone 21-acetate (XVIII),by first selectively protecting the 3-20-diketo groups as describedabove for the mono-enes and then selectively reducing the ll-ketofunction.

CHaOR Sequence A:

IG'pregnene-E a-ol-11,20-di0ne 3-acetate C Ha C 00' I II16-methy1-16-pregnene-3 a-01-l1,20-dione 3-acetatelfifi-methylpregnane-Ii u.-01-11,20-dl0118 3'aeetate III IV165-rnethylpregnane-3 11,17 a-di01-11,20-di0118 lfigimethszlpreguane-i-1,17 a,21-trio1-11,20-dione -ace a e V VIlfifl-methylpregnane-lh,21-dio1-3,11,20-tri0ne4-br0mo-1GB-methylpregnaned7 a,21-di01-3,11,20-

ZI-acetate trione Zl-acetate VII VIII lsp-methylcortisone 21-acetatelfifl methylcortisone IX X lfifl-methylpre dnisone Sequence B:

16B-methyIpregnane-17 a,21-d1o1-3,11,20-tr1one2,4-d1br0mo-16B-methylpregnane-17a,21-diol-3,11,20-

21-acetate trione 21-acetate VII XII lfifi-methylprednisone 21-acetateXIII Sequence 0:

lsfi methylcortisone Zl-acetate lfifl-methylcortlsone8,20bis-sem1carbazone 21-acetate IX XIV lfip-methylhydrocortisone;3,20-bis-semicarbazone lfifl-methylhydrocortisone XV XVIlfifl-methylprednisolone lfifi-methylprednlsolone 21-acetate XVII XVIIIEXAMPLE 1 l6B-methylcortisone 2 l-acetate (A) Product from the reactionof 16-pregnene-3a-ol- 11,20-dione B-acetate and diazomethane.A solutionof 3.72 grams of 16-pregnene-3a-01-11,20-dione 3-acetate in about 1 gramof diazomethane in 70 ml. of ether which had been cooled to -10 C. Themixture is kept at 0 C. for four hours, then allowed to warm up to roomtemperature. The resulting pyrazoline crystallizes directly from thereaction mixture to yield 3.32 g., M.P. 199200 C. dec., [a] +149.6(dioxane).

Analysis.--Calcd. for C H O N C, 69.23; H, 8.27; N, 6.76. Found: C,69.51; H, 7.98; N, 6.69.

(B) 16-methyl-16-pregnene 3a ol-11,20-dione 3-acetate.-The pyrazoline ofExample 1A (3.00 g.) is heated under reduced pressure to about 210 C.until the evolution of nitrogen ceases. The resulting oil is cooled toroom 5 ml. of methylene chloride is added to a solution of 75temperature, then crystallized by the addition of ether.

The precipitate is filtered and dried, yielding 2.12 g. of16-methyl-16-pregnene-3a-ol-11,20-dione 3-acetate, M.P. 163-166 C., [a]|69.9 (dioxane) Amax 248 me (6 10,800).

Analysis.Calcd. for C H O C, 74.57; H, 8.87. Found: C, 74.85; H, 8.55.

(C) 16,8-methylpregnane 3a ol-11,20-dione 3-acetate.-A solution of 12.0g. of the 16-pregnene compound of Example 1B in 250 ml. of glacialacetic acid is hydrogenated at room temperature and atmospheric pressure in the presence of 3.0 g. of 10% palladium on charcoal catalyst.After the reduction is completed (about 1.5 hours), the catalyst isremoved by filtration, and the filtrate concentrated under reducedpressure to about 100 ml. The residue is poured into water, and theprecipitated solid filtered, washed and dried, yielding 11.4 g. ofimpure 16fi-methylpregnane 3a ol-11,20-dione 3-acetate, M.P. 148-155 C.After recrystallization from acetonehexane there is obtained 8.40 g. ofthe product of this example, M.P. 160163 C. no U.V. absorption at 220-300 mn, [a] +93.6 (dioxane).

Analysis.Calcd. for C H O C, 74.19; H, 9.34. Found: C, 74.37; H, 9.06.

(D) 16/3-methylpregnane-3a,17a-diol-l1,20 dione.-A solution of 6.77 g.of 16fi-methylpregnane-3u-ol-l1,20- dione 3-acetate, in 156 ml. ofacetic anhydride containing 3.89 g. of p-toluenesulfonic acid is kept at100 C. for six hours; during which time about 16 ml. of distillate isremoved every half hour by the application of vacuum. The resulting oilyresidue is dissolved in 80 ml. of benzene and washed three times withwater, then with a solution of 1.55 g. of sodium acetate in 20 ml. ofwater. The benzene layer is dried over magnesium sulfate, and thenstirred for 18 hours at 25 C. with a mixture of 0.52 g. of sodiumacetate in 12 ml. of commercial 40% peracetic acid. Excess peraceticacid is then destroyed by the dropwise addition of a solution of 15.5 g.of sodium sulfite in 52 ml. of water, while maintaining the temperaturebetween 10-20 C. An additional 1.57 g. of sodium sulfite is then added,and the mixture stirred overnight until a starch-iodide test isnegative. The benzene layer is separated, washed three times with water,and evaporated. To the resulting residue which is dissolved in 345 ml.of methanol, there is added a solution of 3.62 g. of sodium hydroxide in39.5 ml. of water, and the mixture refluxed for 15 minutes. Afterneutralization with 4 ml. of glacial acetic acid, the solution isconcentrated under reduced pressure to a volume of about 50 ml. Thisconcentrate is poured into a mixture of ice and water and the solidwhich precipitates is filtered and dried, yielding 6.69 g. of impure16,3-methylpregnane-3a-17a-diol-11,20-dione, M.P. 105- 140 C. This waschromatographed on Florisil, and the material eluted with 33-50% etherhexane was combined and crystallized from acetone-hexane, yielding 2.09g. of 16/i-methylpregnane-3e,17a-diol-11,20-dione, M.P. 181.5- 185 C.[t] +83.6 (dioxane).

Analysis.Calcd. for C H O C, 72.89; H, 9.45. Found: C, 72.82; H, 9.25.

16,3 methylpregnane-3a,17a-diol-11,20-dione 3-acetate is prepared byadding to 1 gram of the corresponding 16,8- methylpregnane-diol asolution of 0.5 ml. of acetic anhydride in 2.3 ml. of pyridine. Afterstanding one hour at room temperature, the reaction mixture is pouredinto ice and hydrochloric acid. The resulting solid is filtered, dried,and recrystallized from acetone-hexane to yield 16/3-methylpregnane-3u,17a-diol-11,20-dione 3-acetate, M.P. 169-172 C. Itsinfrared spectrum indicates the presence of an acetate, hydroxyl and twoketonic groups.

Analysis.-Calcd. for C H O C, 71.25; H, 8.97. Found: C, 71.29; H, 8.92.

(E) l6fi-methylpregnane 3a,l7a,2l-tri0l-ll,20 dione 21-acetate.Asolution of 362 mg. of 16/8-methylpregnane-3u,17a-diol-11,20-dione in 18ml. of C.P. Chloroform (containing a few drops of chloroform previouslysaturated with hydrogen bromide) is brominated at -25 C. by the addition(over a three hour period) of 165 mg. of bromine in 10 ml. ofchloroform. After removal of the solvent under reduced pressure, 10 ml.of dimethylformamide and one gram of sodium acetate are added. Themixture is stirred at 60 C. for two hours, allowed to stand overnight atroom temperature, then heated another two hours at 60 C. The mixture isthen poured into water, and the precipitated solid filtered and dried,yielding 370 mg. of impure 16/8-methylpregnane-3a,17u,2l-triol-l1,20-dione 21-acetate, M.P. 192-203 C. Recrystallization fromacetone-hexanegives 280 mg. 'of'th'e product of this example, 'M.P.199-205 -C. The analytical sample, after another recrystallization,melts at 200-205.5 C.

Analysis.-Calcd. fOI' C24H3606: C, H, Found: C, 68.79; H, 8.39.

(F) 16B-methylpregnane-17u,21-diol-3,11,20-trione 21- acetate.A solutionof 180 mg. of 16fl-methylpregnane- 3a,17a,21-triol-11,20-dione21-acetate, in 30 ml. of acetone-water is cooled to 10 C. One drop ofconcentrated hydrochloric acid is added, along with 150 mg. ofN-bromosuccinimide, and the mixture allowed to react 22 hours at 10 C.in the dark. Excess sodium sulfite solution is then added, and themixture concentrated under reduced pressure to a small volume from whichprecipitates 120 mg. of impure 16/3-methylpregnane-17a,21-diol-3,11,20-trione 2l-acetate, M.P. 190-195 C. dec. Purification is effected bymeans of zinc dust in aqueous acetone containing 2 drops of acetic acid.The zinc is removed by filtration, the filtrate concentrated, and hotwater added to crystallize 100 MG. of the product of this example, M.P.198-202 C.

(G) 4B-bromo-1fifi-methylpregnane-lhfl1-diol-3,11,20- trione21-acetate.A solution of 84 mg. of16,8-methylpregnane-17a,2l-diol-3,l1,20-trione 21-acetate in 3-ml. oftert.-butyl alcohol and 3 ml. of methylene chloride is brominated at30-35 C. by the rapid addition of a solution of 32 mg. of bromine in 3ml. of tert.-butyl alcohol. After bromination is complete (1% hours),the solution is evaporated to dryness under reduced pressure. Theresidue is slurried with water, filtered and dried to yield mg. of4,19-bromo-165-methylpregnane-17a,21-di0l-3,11, 20-trione 2l-acetateM.P. 115130 C. dec., which is of suflicient purity for the conversiondescribed in the following procedure.

(H) 16B-methylcortisone 21-acetate.The 4,8-bromo-16,8-methylpregnane-17a,21-diol-3,1 1,20-trione 21-acetate of aboveExample 1G is dissolved in a mixture of 30 mg. of semicarbazide, 4 ml.of tert.-butyl alcohol and 2 ml. of methylene chloride, and stir-red ina nitrogen atmosphere for two hours at 25-30 C. After removing thesolvent under reduced pressure, the residue is dissolved in 5 ml. of 80%acetic acid-water along with 70% excess pyruvic acid, and the reactionmixture allowed to stand at room temperature for 20 hours. The solutionis brought to about pH 7 with dilute (5%) sodium hydroxide, and then isextracted with methylene chloride. The organic extracts are evaporatedto a residue, and the residue chromatographed on Florisil. Crystallinematerial (25 mg.) obtained from the 50% ether-hexane and ether eluates,on crystallization from acetone-hexane yields 20 mg. oflfifl-methylcortisone 21-acetate, M.P. 198-208 C.

max.

EXAMPLE 2 16,8-methylcortisone 9 EXAMPLE 3 16/3-methylhydrocortisone (A)16fi-methylcortisone 21-acetate 3,20-bis-semicarbazone.A mixture of 5.0g. of 1613-rnethylcortisone, 8.3 g. of semicarbazide hydrochloride, 6 g.of pyridine, 50 ml. of water and 200 ml. of methanol is refluxed for 16hours. The solution is concentrated to about 60 ml., then poured intowater to precipitate l6fl-methylcortisone 21- acetate3,20-bis-semicarbazne.

(B) 16,8 methylhydrocortisone 3,20 bis seniicarba- 2one.A solution of6.0 g. of the bis-semicarazone of above Example 3(A) and 4 g. ofpotassium borohydride in 200 ml. of tetrahydrofuran and 100 ml. of wateris refluxed for 6 hours. The solution is cooled, and acetic acid addedto pH 5.5. The organic solvent is distilled and the solids in theresidue are filtered to give 16/3-methylhydrocortisone3,20-bis-semicarbazone.

(C) 16/3-methylhydrocortisone.Under a nitrogen atmosphere, 5.0 g. of thebis-semicarbazone of above Example 3(B) is dissolved in 250 ml. of 2,4 Nhydrochloric acid. The solution is cooled to C., then 2.5 g. of sodiumnitrite in 25 ml. of water is added over a minute period at 5 C. Thereaction mixture is stirred an additional 30 minutes, then cooled tobelow 15 C., neutralized with sodium hydroxide, and extracted severaltimes with chloroform. The solvent is evaporated under reduced pressureto give a solid residue which, after crystallization fromacetone-hexane, yields 16fi-methylhydrocortisone.

EXAMPLE 4 16 8-methylhydrocortisone 21-acetate To one gram of16p-methylhydrocortisone, prepared as in Example 3, is added 0.5 ml. ofacetic anhydride in 2.3 ml. of pyridine. After standing for one hour atroom temperature, the mixture is poured into ice and hydrochloric acid.The resulting precipitate is filtered and recrystallized from aqueousmethanol to yield 16,8-methylhydrocortisone Ill-acetate.

EXAMPLE 5 16/8-methylprednisone ZI-acetate (A) 2,4 dibromo 16Bmethylpregnane 1711,21- diol3,l1,20-trione 21-acetate.--A solution of167 mg. 16B-methylpregnane-17a,21-diol-3,1 1,20-trione 2l-acetate, thecompound of Example1(F), in 3 ml. of dioxane is dibrorninated inpositions 2 and 4 by the rapid addition of 130 mg. of bromine in 1 ml.of dioxane at room temperature. The solution is poured into water andthe precipitated solid is filtered, yielding 180 mg. of2,4-dibromo-l6fi-methylpregnane-l7a,21-diol-3, 1 1,20-trione 21- acetateof sufiicient purity to be used in the conversion described in thefollowing procedure.

(B) 165-methylprednisone 2l-acetate.The 2,4-dibromide (180 mg.) asprepared in above Example 5 (A) is dehydrobrominated by refluxing for 2hours with 4 ml. of dimethylformamide containing 30 mg. of calciumcarbonate. The mixture is poured into dilute hydrochloric acid andextracted with methylene chloride. The organic extract is evaporated toa residue (150 mg), which is chromatographed on Florisil. The fractionsobtained by eluation with 20% ether-hexane are crystallized fromacetone-hexane to give 40 mg. of 16fi-methylprednisone 21-acetate, M.P.2102l6. One further crystallization gave M.P. 215-218",

237 mp (e 10,500)

EXAMPLE 6 16B-methylprednisone 16B-methylprednisone 21-acetate (0.5 g.),the compound of Example 5, when hydrolyzed by means of aqueous alcoholicpotassium bicarbonate in the manner described in Example 2 yields16,8-methy1prednisone.

An alternative method of the preparation of the compound of this exampleis as follows:

Bacillus sphaericus var. fusiformis (A.T.C.C. 7055) is incubated on anutrient agar (composed of Bacto-beet extract, 3 g.; Bacto-peptone, 5g.; sodium chloride, 8 g.; agar, 15 g.; tap water, 1 liter) for 24 hoursat 28 C.

To ml. of a sterile nutrient broth (composed of Bacto-beef extract, 3g.; Bacto-peptone, 5 g.; per liter of tap water) in a 300 ml. flask isadded one loopful of the incubated culture and the broth mixture isfurther incubated for 24 hours at 28 C. on a shaking machine. The brothculture so obtained is employed as in inoculum (1% Into each of tenflasks containing 100 ml. of sterile nutrient broth is added 1 ml. ofthe inoculum. The flasks are agitated on a rotary shaker for 8 hours at28 C. at 240 strokes per minute. After this growth period, a solution of25 mg. of 16,8-methylcortisone, the compound of Example 2, in 0.5 ml. ofmethanol is aseptically added to each flask which in turn is reshakenand incubated for an additional 24 hours. The final pH is 7.8.

The contents of the flasks are then combined and extracted three timeswith 2 liters of chloroform per extraction. The combined chloroformextracts are evaporated to dryness yielding 310 mg. of crude product.The crude steroid is purified by chromatography on a chromatographicsystem described by G. M. Shull, Abstracts of Papers of the 126thMeeting of the American Chemical Society, Dec. 12-17, 1954, page 9a,paper No. 24. Chromatographic evaluation shows a quantitative conversionof the starting material to the diene when an authentic sample of the16,3-methylprednisone is used as a control.

Alternatively, the crude product is recrystallized from acetoneaffording 225 mg. of 16B-methylprednisone.

EXAMPLE 7 16B-methylprednisolone 16,8-methylhydrocortisone, the compoundof Example 3, is fermented by means of Bacillus sphaericus var.fusiformis (A.T.C.C. 7055) in the manner described in the alternateprocedure of Example 6 to produce 166- methylprednisolone.

EXAMPLE 8 16fi-methylprednisolone 21-acetate 16,8-methylprednisolone ofExample 7 is treated with acetic acid and pyridine in the mannerdescribed in Example 4 to yield l6 3-methylprednisolone 21-acetate.

We claim:

1. 3a,20-diacetoxy-l6fi-lower alkyl-l7(20)-pregnene- 1 l-one.

2. 3,a20-diacetoxyl-165-methyl-17(20) pregnene 11- one.

3. 3a,20-diacetoxy-16,i'-lower alkyl-17(20)-oxido-pregnane-l l-one.

4. 3 u,20-diacetoxy-16,8-methyl-17 (20) -oxido-pregnanell-one.

References Cited UNITED STATES PATENTS 2,790,799 4/1957 Djerassi260-23955 2,932,656 4/1960 Day 260-397.45

FOREIGN PATENTS 736,182 9/1955 Great Britain. 741,732 12/1955 GreatBritain. 748,914 5/ 1956 Great Britain.

OTHER REFERENCES Mueller et al.: Bull. Soc. Chim., France (1956), pp.1457-9 relied on.

ELBERT L. ROBERTS, Primary Examiner US. Cl. X.R.

11-1050 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.5, 117 Dated August 1 1969 Inventor) Richard Rausser and Eugene P.Oliveto It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

r- Column 5, line 16, "pertifluoroacetic" should read--pertrifluoroacetic--. Column 4, line 20, "lGB-methylpregnz l70.'.2l-"should read --l6B-methylpregnane-l70z, 21-

Column h, line 57, "the recations" should. read q--the reaction:

Column 6, at the top, that portion of structural formula B rear shouldread Column 7, line 51, "-fia-l'b-diol" should read -5oz, l70t-diol-Column 8, line 51 "100 M.G. should read --l0O mg.---. Column 10, line52, claim 2, "5,0120-" should read --30t,20-

SIGNED RND SEALED sum Attest:

Edward M. Fletcher, Ir. WILLIAM um. Attesting Officer communion"? ofPate

3. 3A,20-DIACETOXY-16B-LOWER ALKYL-17(20)-OXIDO-PREGNANE-11-ONE.